J. Agric. Food Chem. 2003, 51, 1771−1776
1771
Establishment and Application of a Fluorescent Polymerase Chain Reaction−Restriction Fragment Length Polymorphism (PCR-RFLP) Method for Identifying Porcine, Caprine, and Bovine Meats YU-LING SUN†
AND
CHICH-SHENG LIN*,§
Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, and Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu 300, Taiwan
A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCRRFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the speciesspecific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy. KEYWORDS: Meat identification; PCR-RFLP; mitochondrial DNA; 12S ribosomal RNA gene
INTRODUCTION
Identifying the species of origin of meat represents a considerable problem for food and animal feed analysts, game law enforcement authorities, and also those seeking to comply with certain religious regulations. The food analyst is confronted with providing proof of fraudulent substitution of more expensive meats with cheaper meats. Identification of the meat species is a prerequisite for the regulatory control of such products. Moreover, recent consumer concern relating to bovine spongiform encephalopathy (BSE) has increased awareness regarding the composition of food products and animal feed. The past decade has seen DNA replace protein in species identification due to its stability at high temperatures and the fact that its structure is conserved within all tissues of an individual (1). DNA-based methods are based on the presence of species-specific sequences of DNA in meat and the possibility of detecting such sequences specifically. This has resulted in the development of species-specific DNA probes (i.e., DNA hybridization) (2-5), Polymerase Chain Reaction (PCR) assays (6-9), random amplified polymorphic DNA (RAPD) (10-13), and PCR-restriction fragment length polymorphism (RFLP) (7, 14-16).
PCR-RFLP allows the amplification of a conserved region of DNA sequence using PCR and the detection of genetic variation among species by digestion of the amplified fragment with restriction enzymes. This technique has been used for speciation by exploiting DNA sequence variation within the mitochondrial DNA (mtDNA) including the D-loop region (17) and cytochrome b (Cytb) gene (14, 16). Meyer et al. (14) have applied the PCR-RFLP analytical method based on mitochondrial Cytb to the detection of pork in both fresh and heated mixtures of pork and beef at sensitivity levels of
